CN103267854B - A kind of method strengthening detection paper signal - Google Patents

Abstract

A method for enhancing the detection signal of test paper, first preparing blank lateral flow test paper strips, then selecting or preparing indicator particles and enhancing particles, and then determining capture molecules, detection molecules, control molecules, first enhancing molecules and second enhancing molecules, The detection molecule and the control molecule are respectively configured into a detection solution and a control solution, and added to the test paper chromatography membrane to form a detection area and a control area, and the granular polymer is prepared, and the prepared granular polymer is added to the test paper binding pad as a color development Particles, dry the binding pad, take the dried test paper strip, soak one end of the binding pad in the sample to be tested, keep the solution out of contact with the granular polymer, after the absorbent pad is partially wet, take out the test paper, observe and detect Both the detection area and the control area develop color, indicating that the result is positive; the detection area does not develop color, but the control area develops color, indicating that the result is negative; the control area does not develop color, the result is unreliable, and repeated testing is required. The invention is simple and versatile.

Description

A method for enhancing the detection signal of test paper

technical field

The invention belongs to the technical field of biological test paper detection, and in particular relates to a method for enhancing test paper detection signals.

Background technique

At present, there are mainly four types of biochemical analysis devices developed based on test paper, namely dip-dye test paper (such as pH test paper), lateral flow test paper (such as early pregnancy test paper), longitudinal flow test paper (1. OhYK, Joung H-A, Kim S, Kim M-G. Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay. Lab on a Chip2013;13(5):768-72) and microfluidic test paper (2. Vella SJ, Beattie P, Cademartiri R, Laromaine A , Martinez AW, Phillips ST, Mirica KA, Whitesides GM. Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Analytical Chemistry 2012;84(6):2883-91). Although the materials used by these four test papers, the assembled structures and the methods used are different, their basic working principle is the same, that is, to convert chemical signals into optical or electrical signals that are easy to detect, and by detecting these signals, the target is detected. Qualitative or quantitative detection of objects. Compared with other signals, the color reaction based on detection of optical signals is simpler, more convenient, and more mature, so it is the most widely used.

At present, there are two methods for converting chemical signals into optical signals, namely direct method and indirect method. The so-called direct method, the principle is that the detection molecule can interact with the target and cause a color change. This method is mainly used for dip-dye test paper and microfluidic test paper. The indirect method is to introduce chromogenic particles as reaction indicators when the detection molecule can interact with the target but does not cause a color change. This method is mainly used for lateral flow test paper and longitudinal flow test paper. For the indirect method, its detection sensitivity is not only affected by the properties of the target itself, but also largely depends on the preparation and application of the chromogenic particles.

At present, there are mainly two methods to improve the detection sensitivity of the indirect method of test paper. One method uses a two-step reaction, that is, a secondary reaction such as enzyme catalysis or silver staining is performed on the basis of the chromogenic particle indicating reaction to further enhance the signal; One method is to improve the indicating ability of chromogenic particles, that is, to use particle polymers instead of traditional single particles as reaction indicators. The former increases the complexity of test paper detection, while the latter retains the simplicity of test paper detection, but has higher requirements for the preparation of particle aggregation, so its application is limited.

Contents of the invention

In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for enhancing the detection signal of the test paper, which is simple and universal.

To achieve the above object, the present invention adopts the following technical solutions:

A method for enhancing test paper detection signal, comprising the following steps:

1) To prepare a blank lateral flow test strip, first paste the binding pad, chromatographic membrane, and absorbent pad on the support pad in sequence, with the chromatographic membrane in the middle, the binding pad and the absorbent pad at both ends, and the binding pad and absorbent pad Both overlap with the chromatographic membrane by 1-5mm, and then cut it into test strips with a width of 2-8mm.

The binding pad is used to carry color-developing particles, and the material is glass fiber membrane or polyester fiber membrane; the chromatographic membrane is used to fix detection molecules and reference molecules, and the material is nylon membrane, polyester membrane, PVDF membrane, cellulose acetate plain film or nitrocellulose membrane; the absorbent pad is used to absorb the reaction solution and provide driving force for the flow of the test paper solution, and the material is cellulose, cotton wool or cotton fiber; the support pad is used as a support to provide the test paper Mechanical strength, the material is polyvinyl chloride or polystyrene;

2) Select or prepare indicator particles and enhancement particles, the particle size of the indicator particles is 10N nm, used to indicate the detection signal of the test paper, capable of natural color development or chemiluminescence, specifically including gold, selenium, ferric oxide or rare earth nanoparticles; enhancement The particle size is 10M nm, which is used to enhance the detection signal of the test paper, specifically any one of gold, selenium, ferric oxide and rare earth nanoparticles, where 1≤N≤M≤10;

3) Determine the capture molecule, detection molecule, control molecule, first enhancer molecule and second enhancer molecule. The capture molecule can not only specifically bind to the target, but also bind to the indicator particle; the detection molecule can specifically bind to the target, The control molecule can specifically react with the capture molecule, and both the detection molecule and the control molecule can bind to the chromatographic membrane; the first and second enhancer molecules can specifically bind to each other, and the first enhancer molecule can bind to the indicator particle, and the second enhancer molecule can bind to the chromatographic membrane. Enhanced molecular energy and enhanced particle binding;

4) Use water, TAE buffer, TBE buffer or PBS buffer containing 1% to 10% (v/v) methanol, ethanol or isopropanol as solvent, PBS buffer is 10 to 100mM, pH=7.4, The detection molecule and the control molecule are respectively configured as a detection solution and a control solution, with a concentration of 1 μM to 1 mM, and 0.2 to 0.8 μL of each of the two solutions is added to the test paper chromatography membrane to form a corresponding detection area and a control area. Two areas The distance interval is 1mm ~ 1cm, and the chromatographic membrane is dried;

5) Mix the capture molecule and the first enhancer molecule at a molar ratio of 10N:(1~10), and mix the mixed molecule and 10N nm indicator particles at a molar ratio of (30~60)N ² :1 to form the first particle conjugation material, the second enhancer molecules and 10M nm enhancer particles were mixed at (30-60) M ² : 1 to form the second particle conjugate, and the first particle conjugate and the second particle conjugate were mixed in a molar ratio ( 1-10) M:N mixing to obtain a granular polymer; adding the prepared granular polymer to the test paper binding pad as color-developing particles, and drying the binding pad;

6) Take the dried test strip, soak one end of the binding pad in the sample to be tested, keep the solution out of contact with the granular polymer, after the absorbent pad is partially wet, take out the test strip, observe the detection area and the control line area, Both the detection area and the control area develop color, indicating that the result is positive; the detection area does not develop color, but the control area develops color, indicating that the result is negative; the control area does not develop color, the result is unreliable, and repeated testing is required.

The invention enhances the detection signal of the test paper by increasing the aggregation degree of the indicator particles. Compared with the traditional test paper detection method, its biggest improvement is that by introducing the second particle conjugates, multiple first particle conjugates are linked together, thus increasing the number of chromogenic particles. The specific method is to introduce a pair of specific binding enhancing molecules on the basis of traditional detection test paper, so as to prepare granular polymers. The method is simple, convenient and widely used, and can be used for nucleic acid, protein and cell detection.

Description of drawings

Fig. 1 is a comparison of nucleic acid detection results in Example 1 of the present invention: wherein: Fig. 1 (A) is the detection result of the enhanced nucleic acid test paper; Fig. 1 (B) is the result of the traditional nucleic acid detection test paper.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1: nucleic acid detection

A method for enhancing test paper detection signal, comprising the following steps:

1) To prepare a blank lateral flow test strip, first paste the binding pad, chromatographic membrane and absorbent pad on the support pad in sequence; the chromatographic membrane is in the middle, the binding pad and absorbent pad are at both ends, and the binding pad and absorbent pad are Both overlap with the chromatographic membrane by 2mm, and then cut it into a test strip with a width of 2.5mm. The binding pad is used to carry color-developing particles, and the material is glass fiber membrane; the chromatographic membrane is used to fix detection molecules and reference molecules, and the material is nitrocellulose membrane; the absorption pad is used to absorb the reaction solution, and the test paper The solution flow provides driving force, and the material is cellulose; the support pad is used as a support to provide a certain mechanical strength for the test paper, and the material is polyvinyl chloride;

2) Choose gold nanoparticles with 10nm for both the indicator particles and the enhancement particles, which are prepared by sodium citrate reduction method; the indicator particles are used to indicate the detection signal of the test paper, and the enhancement particles are used to enhance the detection signal of the test paper;

3) Determine the capture molecule, detection molecule, control molecule, first enhancer molecule and second enhancer molecule. The target, capture molecule, detection molecule, control molecule, first enhancer molecule and second enhancer molecule are shown in Table 1. The capture molecule can hybridize with the nucleic acid at the 5' end of the target, and the alkylthiol SH-(CH ₂ ) _6- connected at its 3' end can bind to gold nanoparticles; the detection molecule can hybridize with the nucleic acid at the 3' end of the target, and the control molecule can The capture molecule can hybridize with nucleic acid, and under the irradiation of ultraviolet light, the detection molecule and the control molecule can combine with the chromatographic membrane; Mercapto SH-(CH ₂ ) _3- can combine with gold nanoparticles;

Table 1 Nucleic acid sequence

The above nucleic acid sequences were all produced by Sangong Biological (Shanghai) Co., Ltd. synthesis;

4) Using PBS buffer (10mM, pH=7.4) containing 10% (v/v) ethanol as the solvent, prepare the detection molecule and the control molecule into the detection solution and the control solution respectively, the concentration is 100μM, and each of the two solutions takes 0.5 Add μL on the chromatographic membrane of the test paper, and irradiate it under ultraviolet light for 3 hours to form the corresponding detection area and control area. The distance between the two areas is 2mm, and the chromatographic membrane is dried in the air;

5) Mix the capture molecule and the first enhancer molecule at a molar ratio of 1:1, mix the mixed molecule with the indicator particle at a molar ratio of 40:1 to form the first particle conjugate, and mix the second enhancer molecule with the enhancer particle at a molar ratio Mix 40:1 to form the second particle conjugate, mix the first particle conjugate and the second particle conjugate at a molar ratio of 6:1 to prepare a particle polymer; add the prepared particle polymer to the test paper As the chromogenic particles on the binding pad, dry the binding pad;

6) Take the dried test paper, soak one end of the binding pad in 80 μL of the target solution of different concentrations, keep the solution away from the granular polymer, after the absorbent pad is partially wet, take out the test paper, observe the detection area and the control Line area, the detection results are shown in Figure 1A. The gold nano-conjugate prepared with the same gold nanoparticles and capture molecules was used as a control. The detection results of the traditional test paper are shown in Figure 1B. The detection limit of the enhanced test paper is 0.25nM, which is 2.5 In addition, the color intensity of the enhanced test paper has been significantly improved compared with the traditional test paper. The above results show that the detection signal of the test paper has been enhanced.

Embodiment 2: Nucleic acid detection

A method for enhancing test paper detection signal, comprising the following steps:

1) To prepare a blank lateral flow test strip, first paste the binding pad, chromatographic membrane and absorbent pad on the support pad in sequence; the chromatographic membrane is in the middle, the binding pad and absorbent pad are at both ends, and the binding pad and absorbent pad are Both overlap with the chromatographic membrane by 2mm, and then cut it into a test strip with a width of 2.5mm. The binding pad is used to carry color-developing particles, and the material is glass fiber membrane; the chromatographic membrane is used to fix detection molecules and reference molecules, and the material is nitrocellulose membrane; the absorption pad is used to absorb the reaction solution, and the test paper The solution flow provides driving force, and the material is cellulose; the support pad is used as a support to provide a certain mechanical strength for the test paper, and the material is polyvinyl chloride;

2) Select gold nanoparticles with 10nm indicator particles and gold nanoparticles with 40nm enhanced particles, both of which are prepared by sodium citrate reduction method; the indicator particles are used to indicate the detection signal of the test paper, and the enhanced particles are used to enhance the detection signal of the test paper ;

3) Determine the capture molecule, detection molecule, control molecule, first enhancer molecule and second enhancer molecule. The target, capture molecule, detection molecule, control molecule, first enhancer molecule and second enhancer molecule are shown in Table 2. The capture molecule can hybridize with the nucleic acid at the 5' end of the target, and the alkylthiol SH-(CH ₂ ) _6- connected at its 3' end can bind to gold nanoparticles; the detection molecule can hybridize with the nucleic acid at the 3' end of the target, and the control molecule can The capture molecule can hybridize with nucleic acid, and under the irradiation of ultraviolet light, the detection molecule and the control molecule can combine with the chromatographic membrane; Mercapto SH-(CH ₂ ) _3- can combine with gold nanoparticles;

Table 2 Nucleic acid sequence

The above nucleic acid sequences were all produced by Sangong Biological (Shanghai) Co., Ltd. synthesis;

4) Using PBS buffer (10mM, pH=7.4) containing 10% (v/v) ethanol as the solvent, prepare the detection molecule and the control molecule into the detection solution and the control solution respectively, the concentration is 100μM, and each of the two solutions takes 0.5 Add μL on the chromatographic membrane of the test paper, and irradiate it under ultraviolet light for 3 hours to form the corresponding detection area and control area. The distance between the two areas is 2mm, and the chromatographic membrane is dried in the air;

5) Mix the capture molecule and the first enhancer molecule at a molar ratio of 1:1, mix the mixed molecule with the indicator particle at a molar ratio of 40:1 to form the first particle conjugate, and mix the second enhancer molecule with the enhancer particle at a molar ratio Mix 640:1 to form the second particle conjugate, mix the first particle conjugate and the second particle conjugate at a molar ratio of 16:1 to prepare a particle polymer; add the prepared particle polymer to the test paper As the chromogenic particles on the binding pad, dry the binding pad;

6) Take the dried test paper, soak one end of the binding pad in 80 μL of the target solution of different concentrations, keep the solution away from the granular polymer, after the absorbent pad is partially wet, take out the test paper, observe the detection area and the control line area. The detection result is similar to that of Example 1. Compared with the traditional detection test paper, the color intensity and detection limit of the enhanced test paper have been significantly improved.

Claims (3)

1. strengthen a method for detection paper signal, it is characterized in that, comprise the following steps:

1) blank lateral flow test strips is prepared, first pad, chromatographic film and absorption pad are pasted on support pad in order, wherein chromatographic film is in centre, pad and absorption pad are at two ends, pad, absorption pad all overlap with chromatographic film 1 ~ 5mm, then the test strips that 2 ~ 8mm is wide is cut into

Described pad is in order to carrying colour developing particle, and material is glass fibre membrane or dacron film; Described chromatographic film is in order to fixed test molecule and contrast molecule, and material is nylon membrane, polyester film, pvdf membrane, cellulose acetate membrane or nitrocellulose filter; Described absorption pad is in order to absorption reaction liquid, and provide driving force to the flowing of test paper solution, material is cellulose, cotton young suede or cotton fiber; Described support pad is used as holder, and provide mechanical strength to test paper, material is Polyvinylchloride or polystyrene;

2) to select or preparation instruction particle and strengthen particle, the granularity of instruction particle is 10N nm, is used to indicate detection paper signal, can natural colour developing or chemiluminescence, specifically comprises gold, selenium, tri-iron tetroxide or rare earth nanometer particle; The granularity strengthening particle is 10M nm, for strengthening detection paper signal, is specially any one in gold, selenium, tri-iron tetroxide and rare earth nanometer particle, wherein 1≤N≤M≤10;

3) determine capture molecules, detection molecules, contrast molecule, first strengthen molecule and second strengthen molecule, capture molecules can not only with object specific binding, and can and instruction particle combine; Detection molecules energy and object specific binding, contrast molecular energy and capture molecules specific reaction combine, and detection molecules and contrast molecule can combine with chromatographic film; First, second strengthens the mutual specific binding of molecular energy, and first strengthens molecular energy and indicate particle to combine in addition, and second strengthens molecular energy and strengthen particle combination;

4) with containing the water of 1% ~ 10% (v/v) methyl alcohol, ethanol or isopropyl alcohol, TAE damping fluid, tbe buffer liquid or PBS damping fluid for solvent, PBS damping fluid is 10 ~ 100mM, pH=7.4, detection molecules and contrast molecule are configured to detect solution and contrast solution respectively, concentration is 1 μM ~ 1mM, respectively gets two kinds of solution 0.2 ~ 0.8 μ L and is added in test paper chromatographic film, form corresponding detection zone and check plot, two region distances are spaced apart 1mm ~ 1cm, chromatographic film are dried;

5) capture molecules and first are strengthened molecule 10N:(1 ~ 10 in molar ratio) mixing, by instruction particle (30 ~ 60) N in molar ratio of mixed molecules and 10N nm ²: 1 is mixed to form the first particle conjugate, strengthens the enhancing particle of molecule and 10M nm by (30 ~ 60) M by second ²: 1 is mixed to form the second particle conjugate, by the first particle conjugate and the second particle conjugate in molar ratio (1 ~ 10) M:N mix, obtained particulate polymers; Obtained particulate polymers is added on test paper pad as colour developing particle, pad is dried;

6) get dried test strips, its pad one end is infiltrated in testing sample, keep solution not contact with particulate polymers, after pad partially wetting to be absorbed, take out test paper, observe detection zone and control line district, all developing the color in detection zone and check plot, shows that result is positive; Not developing the color in detection zone, develops the color in check plot, shows that result is negative; Do not develop the color in check plot, result is unreliable, needs duplicate detection.

2. a kind of method strengthening detection paper signal according to claim 1, is characterized in that, comprise the following steps:

1) prepare blank lateral flow test strips, first pad, chromatographic film and absorption pad are pasted on support pad in order; Wherein chromatographic film is in centre, and pad and absorption pad are at two ends, and pad, absorption pad all overlap with chromatographic film 2mm, are then cut into the test strips that 2.5mm is wide, and described pad is in order to carrying colour developing particle, and material is glass fibre membrane; Described chromatographic film is in order to fixed test molecule and contrast molecule, and material is nitrocellulose filter; Described absorption pad is in order to absorption reaction liquid, and provide driving force to the flowing of test paper solution, material is cellulose; Described support pad is used as holder, and provide certain mechanical strength to test paper, material is Polyvinylchloride;

2) select instruction particle and strengthen the nanogold particle that particle is 10nm, being obtained by reduction of sodium citrate method; Instruction particle is used to indicate detection paper signal, strengthens particle for strengthening detection paper signal;

3) determine capture molecules, detection molecules, contrast molecule, first strengthens molecule and the second enhancing molecule, object, capture molecules, detection molecules, contrast molecule, first strengthen molecule and the second enhancing molecule is as shown in table 1, capture molecules energy and object 5 ' hold nucleic acid hybridization, the alkane sulfydryl SH-(CH that its 3 ' end connects ₂) ₆-can be combined with gold nano grain; Detection molecules energy and object 3 ' hold nucleic acid hybridization, contrast molecular energy and capture molecules nucleic acid hybridization, under UV-irradiation, and detection molecules and contrast molecular energy and chromatographic film combination; First, second strengthens the mutual cross of molecular energy phase, and first, second strengthens the alkane sulfydryl SH-(CH that molecule 5 ' holds connection ₂) ₃-energy and gold nano grain combine;

Table 1 nucleotide sequence

Above-mentioned nucleotide sequence is by raw work the synthesis of biological (Shanghai) Co., Ltd.;

4) to contain 10% (v/v) ethanol PBS damping fluid for solvent, ethanol PBS damping fluid is 10mM, pH=7.4, detection molecules and contrast molecule are configured to respectively detect solution and contrast solution, concentration is 100 μMs, respectively gets two kinds of solution 0.5 μ L and is added in test paper chromatographic film, and irradiate 3h under ultraviolet light, form corresponding detection zone and check plot, two region distances are spaced apart 2mm, chromatographic film are dried;

5) capture molecules and first are strengthened molecule 1:1 mixing in molar ratio, by mixed molecules and instruction particle in molar ratio 40:1 be mixed to form the first particle conjugate, by second strengthen molecule with strengthen particle in molar ratio 40:1 be mixed to form the second particle conjugate, by the first particle conjugate and the 6:1 mixing in molar ratio of the second particle conjugate, obtained particulate polymers; Obtained particulate polymers is added on test paper pad as colour developing particle, pad is dried;

6) get dried by test paper, its pad one end is infiltrated in the object solution of 80 μ L variable concentrations, solution is kept not contact with particulate polymers, after pad partially wetting to be absorbed, take out test paper, observe detection zone and control line district, the nm of gold conjugate obtained with identical gold nano grain and capture molecules in contrast, detection is limited to 0.25nM, and the detection strengthening test paper is limited to 0.1nM, than improve 2.5 times.

3. a kind of method strengthening detection paper signal according to claim 1, is characterized in that, comprise the following steps:

1) prepare blank lateral flow test strips, first pad, chromatographic film and absorption pad are pasted on support pad in order; Wherein chromatographic film is in centre, and pad and absorption pad are at two ends, and pad, absorption pad all overlap with chromatographic film 2mm, are then cut into the test strips that 2.5mm is wide, and described pad is in order to carrying colour developing particle, and material is glass fibre membrane; Described chromatographic film is in order to fixed test molecule and contrast molecule, and material is nitrocellulose filter; Described absorption pad is in order to absorption reaction liquid, and provide driving force to the flowing of test paper solution, material is cellulose; Described support pad is used as holder, and provide certain mechanical strength to test paper, material is Polyvinylchloride;

2) select instruction particle to be the nanogold particle of 10nm, strengthening particle is the nanogold particle of 40nm, and nanogold particle obtains by reduction of sodium citrate method; Instruction particle is used to indicate detection paper signal, strengthens particle for strengthening detection paper signal;

3) determine capture molecules, detection molecules, contrast molecule, first strengthens molecule and the second enhancing molecule, object, capture molecules, detection molecules, contrast molecule, first strengthen molecule and the second enhancing molecule is as shown in table 2, capture molecules energy and object 5 ' hold nucleic acid hybridization, the alkane sulfydryl SH-(CH that its 3 ' end connects ₂) ₆-can be combined with gold nano grain; Detection molecules energy and object 3 ' hold nucleic acid hybridization, contrast molecular energy and capture molecules nucleic acid hybridization, under UV-irradiation, and detection molecules and contrast molecular energy and chromatographic film combination; First, second strengthens the mutual cross of molecular energy phase, and first, second strengthens the alkane sulfydryl SH-(CH that molecule 5 ' holds connection ₂) ₃-energy and gold nano grain combine;

Table 2 nucleotide sequence

Above-mentioned nucleotide sequence is by raw work the synthesis of biological (Shanghai) Co., Ltd.;

4) to contain 10% (v/v) ethanol PBS damping fluid for solvent, ethanol PBS damping fluid is 10mM, pH=7.4, detection molecules and contrast molecule are configured to respectively detect solution and contrast solution, concentration is 100 μMs, respectively gets two kinds of solution 0.5 μ L and is added in test paper chromatographic film, and irradiate 3h under ultraviolet light, form corresponding detection zone and check plot, two region distances are spaced apart 2mm, chromatographic film are dried;

5) capture molecules and first are strengthened molecule 1:1 mixing in molar ratio, by mixed molecules and instruction particle in molar ratio 40:1 be mixed to form the first particle conjugate, by second strengthen molecule with strengthen particle in molar ratio 640:1 be mixed to form the second particle conjugate, by the first particle conjugate and the 16:1 mixing in molar ratio of the second particle conjugate, obtained particulate polymers; Obtained particulate polymers is added on test paper pad as colour developing particle, pad is dried;

6) get dried by test paper, its pad one end is infiltrated in the object solution of 80 μ L variable concentrations, solution is kept not contact with particulate polymers, after pad partially wetting to be absorbed, take out test paper, observe detection zone and control line district, the colored intensity and the detectability that strengthen test paper are all significantly improved.